Ribonucleotide reductases catalyze the conversion of nucleotides to deoxynucleotides in all organisms supplying the monomeric precursors required for DNA replication and repair. The class Ia and Ib RNRs are composed of two subunits (? and ) which form the active (?2)n2 complex. 2 houses the essential dinuclear-tyrosyl radical (Y ) cofactor which in the class Ia RNRs from E. coli, S. cerevisiae, and humans is a diferric cluster (FeIII2-Y ), and in the class Ib RNRs from E. coli, B. subtilis, S. sanguinis and many pathogenic microorganisms is likely, a dimanganese cluster (MnIII2- Y ). We are interested in how these cofactors are biosynthesized and repaired, i.e., if the Y is reduced inside the cell by endogenous reductants or the chemotherapeutic hydroxyurea, how the MeIII2-cluster is re-converted to active cofactor. Studies of self-assembly of FeIII2-Y in the Ia RNRs in vitro have demonstrated the requirement for reducing equivalents, and controlled metal and oxidant delivery. We have recently discovered in E. coli, a 2Fe2S cluster ferredoxin (YfaE) that plays a role in electron transfer in cluster biosynthesis and maintenance and likely is involved in cluster metallation. In assembly of the eukaryotic class Ia cluster in S. cerevisiae, Dre2 and Tah18 are proposed to play a similar role in electron transfer for biosynthesis and maintenance and Grx3/Grx4 and Dre2/Tah18 are proposed to play a role in metallation. While the class Ib RNRs can also self-assemble FeIII2-Y active in nucleotide reduction, our recent studies suggest that the active cofactor in vivo is MnIII2-Y cluster and that its assembly in vitro and in vivo require an unusual flavodoxin, NrdI. The present proposal is focused on obtaining evidence for the roles of the protein factors in vivo (E. coli and S. cerevisiae) using the tools we have developed over the last few years (whole cell EPR, antibodies, activity assays, isogenic strains with gene deletions and conditional expression and X-ray crystallography). We are also investigating the mechanism of MnIII2-Y cofactor assembly using time resolved biophysical methods (stopped flow, rapid freeze quench EPR and EXAFS spectroscopies). The observation of the long ago postulated, but elusive, Mn-RNR raises the importance of the competition between pathogens and their hosts for controlled metallation and suggests new targets for antibacterial therapeutics.